The ability of proteins to respond to the transient presence of charge in their interior is critical for their biological function. This is what governs energy transduction, and it is also critical in catalysis. Despite the universal importance of this functional property, the manner in which charge is accommodated in the protein interior, the nature of the structural response to the ionization of an internal group, and the molecular determinants of the pKa values of internal residues, remain unknown. Experimental studies are proposed to characterize, at an unprecedented level of detail, the structural and energetic consequences of ionization processes inside proteins. 50 variants of staphylococcal nuclease, engineered previously in our laboratory with Lys or Glu in 25 different internal positions, will be used for this purpose. X-ray crystallography, NMR spectroscopy, and equilibrium thermodynamic experiments will be used to characterize static and dynamic responses, triggered by the ionization of internal residues. The roles of permanent and induced dipoles, of interactions between interior and surface charges, of water penetration, and of local unfolding will be studied. Computational methods will also be brought to bear on the problem, as necessary. For example, in collaboration with other groups, local polarizabilities will be calculated with quantum methods, standard molecular dynamics (MD) will be used to study water penetration and dipolar relaxation, and replica-exchange MD methods will be used to study how ionization of internal groups can rearrange the backbone. The experimental data obtained from these studies will be used to refine and to test the performance of the most popular computational methods for structure-based pKa calculations. The physical insight that will emerge from the combined experimental and computational studies will further understanding of a key functional motif that is central to all living systems. This could impact our understanding of the molecular basis of many diseases, and it will improve our ability to design drugs and proteins rationally.